Caki-1 and 786-O, which are human RCC cell lines with high and low metastatic potential, respectively, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Caki-1 cells were cultured in McCoy’s 5A medium (Gibco, Grand Island, NY, U.S.) supplemented with 15 % fetal bovine serum (FBS; Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., Shanghai, China), and 786-O cells were cultured in RPMI 1640 (Wisent, Saint-Jean-Baptiste, Canada) supplemented with 10 % FBS.
Clinical sample collection
Human kidney specimens were obtained from 63 patients who underwent radical nephrectomy for localized clear cell RCC at the General Hospital of Jinan Military Command in China between 2008 and 2013. The collection and use of the samples were reviewed and approved by the Institutional Ethics Committee of General Hospital of Jinan Military Command, and expedited pathological diagnosis and staging of these specimens were performed prior to sampling and transporting them for research. Histological diagnosis was established according to the guidelines of the World Health Organization . Cases were selected according to tissue availability and were not stratified for any known preoperative or pathological prognostic factor. Clinical follow-up data was available for all patients. The median follow-up period for all cases was 37 months (range, 7–65 months). Under the supervision of an experienced pathologist, 63 renal cancer tissue samples were collected (before any treatment was begun) from surgically resected kidneys and immediately stored in liquid nitrogen until RNA or protein extraction.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, U.S.) according to the manufacturer’s protocol. The expression of miR-96 was measured using the Hairpin-it™ miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) with the following primers: Sense 5′-TTTGGCACTAGC ACAT-3′; antisense 5′-GAGCAGGCTGGAGAA-3′. The miRNA synthetic standard in the kit was used as a positive control, according to the manufacturer’s instructions. U6 small nuclear RNA was used as an internal control, with the following primers: Sense 5′-ATTGGAACGATACAGAGAAGAT-3′; antisense 5′-GGAACGCTTCACGAATTT-3′ (GenePharma, Shanghai, China). The relative expression of miR-96 in tissues and cell lines were calculated by the 2-Δct method.
Caki-1 and 786-Ocells were transiently transfected with miR-96 inhibitor, miR-96 mimic and miR-control RNA using Lipofectamine 2000 (Invitrogen). Inhibitor of miR-96 (sequence: 5′-GCAAAAAUGUGCUAGUGCCAAA-3′), mimic of miR-96 (sequence: 5′-UUUGGCACUAGCACAUUUUUGC-3′) and negative miR-control (sequence: 5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from GenePharma. The negative miR-control sequence was non-homologous to any human genomic sequence in order to eliminate potential non-sequence-specific effects as previously reported . RCC cells were seeded in six-well plates and transfected with 4 nM of miR-96 inhibitor, miR-96 mimic or miR-control.
Wound healing assay
Caki-1 and 786-O cells transfected with miR-96 mimic, inhibitor or miR-control were cultured as monolayers, synchronized by starving the cells for 24 h in Dulbecco’s Modified Eagle Medium (Gibco) containing 0.1 % FBS, and wounded by removing a wide strip (approximately 300 μm) of cells across the well with a standard 200 μl pipette tip. Wounded monolayers were washed twice to remove nonadherent cells. Wound healing was monitored by phase-contrast microscopy after 24 h culture in 1 % FBS, quantified using Image J software and expressed as the mean percentage of the remaining cell-free area compared with the area of the initial wound.
The invasive ability of Caki-1 and 786-O cells transfected with miR-96 mimic, inhibitor or miR-control was determined using Matrigel (BD Pharmingen, San Diego, CA, U.S.)-coated 24-well Transwell chambers, with upper and lower culture compartments separated by polycarbonate membranes with 8-μm pores (Corning, Costar, New York City, U.S.). The bottom chamber was filled with RPMI-1640 medium containing 10 % FBS for 786-Ocells or McCoy’s5A medium with 15 % FBS for Caki-1 cells. The transfected cells (1 × 105) were seeded on the top chamber and incubated at 37 °C with 5 % CO2. After 40 h, the cells were removed from the upper surface by scrubbing with a cotton swab, and cells that had migrated to the underside of the membrane were stained with Giemsa (Sigma-Aldrich Co., St. Louis, MO, U.S.). A total of five high-power fields were counted by two independent blinded pathologists, and the mean number of cells per field was calculated. The experiments were performed in triplicate.
To further investigate the role of Ezrin in the inhibition of invasion by miR-96, Caki-1 and 786-O cells were pretreated for 30 min with 10 μM NSC668394, a small molecule inhibitor of Ezrin , followed by transfection with miR-control or miR-96 inhibitor for 24 h. Invasive ability was then examined by Transwell assay as described above.
Western blot analysis
Ezrin expression was assessed by western blotting using standard protocols. Briefly, equal amounts of extracted protein, as determined by Bradford protein assay (Bio-Rad, Hercules, CA, U.S.), were separated by sodium dodecyl sulfate (SDS)-8 % polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, England). Membranes were probed with Ezrin antibody (Abcam, Cambridge, MA, U.S.). After incubation with peroxidase-coupled secondary antibodies (Abcam), blots were developed using enhanced chemiluminescence reagents and exposed to X-ray films to detect labeled proteins. Membranes were then stripped with Re-Blot Plus (Millipore, Billerica, MA, U.S.) and subsequently reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam) as a loading control.
Negative and positive controls were routinely incorporated for quality control in all the above assays. All analyses were performed using SPSS 13 software. Repeated-measures analysis of variance (ANOVA) tests were used to compare multiple groups and p values <0.05 were considered statistically significant. Results are expressed as mean ± standard deviation.